3D imaging of live cells with ultraviolet radiation

ABSTRACT

A method for 3D imaging of cells in an optical tomography system includes moving a biological object relatively to a microscope objective to present varying angles of view. The biological object is illuminated with radiation having a spectral bandwidth limited to wavelengths between 150 nm and 390 nm. Radiation transmitted through the biological object and the microscope objective is sensed with a camera from a plurality of differing view angles. A plurality of pseudoprojections of the biological object from the sensed radiation is formed and the plurality of pseudoprojections is reconstructed to form a 3D image of the cell.

TECHNICAL FIELD

The present invention relates to optical tomographic imaging systems ingeneral, and, more particularly, to optical projection tomography for 3Dmicroscopy, in which a small object, such as a biological cell, isilluminated with ultraviolet radiation for pseudoprojection imaging andreconstruction into a 3D image.

BACKGROUND

Advances in imaging biological cells using optical tomography have beendeveloped by Nelson as disclosed, for example, in U.S. Pat. No.6,522,775, issued Feb. 18, 2003, and entitled “Apparatus and method forimaging small objects in a flow stream using optical tomography,” thefull disclosure of which is incorporated by reference. Furtherdevelopments in the field are taught in Fauver et al., U.S. patentapplication Ser. No. 10/716,744, filed Nov. 18, 2003 and published as USPublication No. US-2004-0076319-A1 on Apr. 22, 2004, entitled “Methodand apparatus of shadowgram formation for optical tomography,” (Fauver'744) and Fauver et al., U.S. patent application Ser. No. 11/532,648,filed Sep. 18, 2006, entitled “Focal plane tracking for opticalmicrotomography,” (Fauver '648) the full disclosures of which are alsoincorporated by reference.

Processing in an optical tomography system begins with specimenpreparation. Typically, specimens taken from a patient are received froma hospital or clinic and processed to remove non-diagnostic elements,fixed and then stained. Stained specimens are then mixed with an opticalgel, inserted into a microcapillary tube and images of objects, such ascells, in the specimen are produced using an optical tomography system.The resultant images comprise a set of extended depth of field imagesfrom differing perspectives called “pseudoprojection images.” The set ofpseudoprojection images can be reconstructed using backprojection andfiltering techniques to yield a 3D reconstruction of a cell of interest.The ability to have isometric or roughly equal resolution in all threedimensions is an advantage in 3D tomographic cell imaging, especiallyfor quantitative image analysis.

The 3D reconstruction then remains available for analysis in order toenable the quantification and the determination of the location ofstructures, molecules or molecular probes of interest. An object such asa biological cell may be labeled with at least one stain or taggedmolecular probe, and the measured amount and location of this biomarkermay yield important information about the disease state of the cell,including, but not limited to, various cancers such as lung, breast,prostate, cervical, stomach and pancreatic cancers.

The present disclosure allows an extension of optical projectiontomography to live cell imaging and is expected to advance cellanalysis, drug development, personalized therapy, and related fields.Until now, live cell microscopy has traditionally been done bynon-labeling 2D imaging techniques such as phase contrast, DIC, andpolarization contrast microscopy.

Native absorbance and fluorescence imaging using deep ultraviolet (DUV)at 250 nm to 290 nm wavelengths has been technically challenging andcauses phototoxicity in irradiated cells. More recently, vital stainshave been used that typically emit fluorescence signals for 3D live cellimaging, because commercial microscopes (of confocal, deconvolution, andmultiphoton excitation varieties) rely on fluorescence for building upmultiple planar slices for generating 3D images. However, in thesecases, the 3D image resulting from a stack of 2D images has about fourtimes less axial resolution as the lateral resolution within each slice,thereby making quantitative analysis imprecise. The ability to haveisometric or roughly equal resolution in all three dimensions is asignificant advantage in 3D tomographic cell imaging, especially forquantitative image analysis.

One advantage of using DUV illumination for live cells is that nativeDNA and protein absorb the light at 260 nm and 280 nm, respectively,without the use of any photochemical label that must permeate the cellmembrane and sometimes the nuclear membrane of the cell, which is in anon-normal state. Furthermore, the label or stain is only anintermediary step toward the measurement of target protein or nucleotide(DNA) which adds a large degree of variability in this measurement.Elimination of such exogenous species would potentially improve theaccuracy of a quantitative measure of protein or nucleotide (DNA), aswell as reduce time, effort and complexity by eliminating steps in thesample preparation. Unfortunately, the use of DUV illumination hasdemonstrated phototoxicity in the past, due to the high dose ofradiation required to stimulate a strong signal.

Recently, however, DUV imaging of live cultured human and mouse cellswas demonstrated at 260 nm and 280 nm using DUV light-emitting diodes(LEDs) (See, for example, Zeskind, B J, et al., “P. Nucleic acid andprotein mass mapping by live cell deep ultraviolet microscopy,” NatureMethods 4(7):567-569 (2007)).

The present disclosure describes a new, novel and surprisingly effective3D imaging system that provides solutions to long felt needs in thefield of DUV 3D imaging of cells, and more particularly, live cells.

BRIEF SUMMARY OF THE DISCLOSURE

A method for 3D imaging of cells in an optical tomography system isprovided including moving a biological object relatively to a microscopeobjective to present varying angles of view. The biological object isilluminated with optical radiation having a spectral bandwidth limitedto wavelengths between 150 nm and 390 nm. Radiation transmitted through,scattered by, or secondarily emitted by the biological object andcaptured by the microscope objective is sensed with a camera to recordimages from a plurality of differing view angles. A plurality ofpseudoprojection images of the biological object from the sensedradiation is formed and the plurality of pseudoprojections isreconstructed to form a 3D image of the cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically shows an example of a system for 3D imaging ofcells in an optical tomography system employing ultraviolet radiation.

FIG. 2 schematically shows an alternate example of a system for 3Dimaging of cells in an optical tomography system with ultravioletradiation using a UV camera and optional adaptive optics.

FIG. 3 schematically shows an embodiment of a temperature-controlledhousing for use in an optical tomography system.

FIG. 4 schematically shows a side view of an example of a microfluidicscartridge as used in a raceway configuration for imaging cells.

FIG. 5 schematically shows a top view of an example of a microfluidicscartridge as used in a racetrack configuration for imaging cells.

FIG. 6 schematically shows an optical tomography process includingseparate imaging stages along the same pathway.

In the drawings, identical reference numbers identify similar elementsor components. The sizes and relative positions of elements in thedrawings are not necessarily drawn to scale. For example, the shapes ofvarious elements and angles are not drawn to scale, and some of theseelements are arbitrarily enlarged and positioned to improve drawinglegibility. Further, the particular shapes of the elements as drawn, arenot intended to convey any information regarding the actual shape of theparticular elements, and have been solely selected for ease ofrecognition in the drawings.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following disclosure describes several embodiments and systems forimaging an object of interest. Several features of methods and systemsin accordance with example embodiments of the invention are set forthand described in the Figures. It will be appreciated that methods andsystems in accordance with other example embodiments of the inventioncan include additional procedures or features different than those shownin Figures. Example embodiments are described herein with respect tobiological cells. However, it will be understood that these examples arefor the purpose of illustrating the principals of the invention, andthat the invention is not so limited.

Additionally, methods and systems in accordance with several exampleembodiments of the invention may not include all of the features shownin these Figures. Throughout the Figures, like reference numbers referto similar or identical components or procedures.

Unless the context requires otherwise, throughout the specification andclaims which follow, the word “comprise” and variations thereof, suchas, “comprises” and “comprising” are to be construed in an open,inclusive sense that is as “including, but not limited to.”

Reference throughout this specification to “one example” or “an exampleembodiment,” “one embodiment,” “an embodiment” or various combinationsof these terms means that a particular feature, structure orcharacteristic described in connection with the embodiment is includedin at least one embodiment of the present disclosure. Thus, theappearances of the phrases “in one embodiment” or “in an embodiment” invarious places throughout this specification are not necessarily allreferring to the same embodiment. Furthermore, the particular features,structures, or characteristics may be combined in any suitable manner inone or more embodiments.

Definitions

Generally as used herein the following terms have the following meaningswhen used within the context of optical microscopy processes:

-   -   “Capillary tube” has its generally accepted meaning and is        intended to include transparent microcapillary tubes and        equivalent items with an inside diameter generally of 500        microns or less.    -   “Depth of field” is the length along the optical axis within        which the focal plane may be shifted before an unacceptable        image blur for a specified feature is produced.    -   “Object” means an individual cell, item, thing or other entity.    -   “Pseudoprojection” includes a single image representing a        sampled volume of extent larger than the native depth of field        of the optics. The concept of a pseudoprojection is taught in        Fauver '744.    -   “Specimen” means a complete product obtained from a single test        or procedure from an individual patient (e.g., sputum submitted        for analysis, a biopsy, or a nasal swab). A specimen may be        composed of one or more objects. The result of the specimen        diagnosis becomes part of the case diagnosis.    -   “Sample” means a finished cellular preparation that is ready for        analysis, including all or part of an aliquot or specimen.        With respect to imaging of live cells, several assumptions are        made in this disclosure: (1) submicron isometric resolution is        required of the chromatin structure in the nucleus which limits        the wavelength of optical radiation to frequencies higher than        infrared (less than or equal to near infrared wavelengths, <1000        nm), (2) individual cells are being imaged or possibly analyzed        which may allow for diffraction measurement at multiple        perspectives, and (3) harvesting of the cell after imaging is        desired with minimal cell damage.

Referring now to FIG. 1 a system for 3D imaging of cells in an opticaltomography system 11 employing ultraviolet radiation is schematicallyshown. A tube 22, such as a capillary tube, microcapillary tube orequivalent, is positioned to be viewed by a microscope 16 including amicroscope objective 18 and a tube lens element 52. A rotationmechanism, for example, a rotary motor 20 is attached to the tube 22. Anaxial translation mechanism, for example motor 34, is coupled to themicroscope objective. A radiation source 29 is positioned to illuminatea part of the tube 22 including a biological object 1 held therein. Theradiation source 29 generates radiation having a spectral bandwidthlimited to wavelengths between 150 nm and 390 nm. In one useful example,the radiation source 29 comprises multiple sources 30, 31 transmittingat least two selected wavelengths that are detected concurrently by afirst light detector 10 and a second light detector 14. Optional filters12A, 12B are selected to block fluorescence having a wavelength longerthan the UV limited spectral bandwidth, such as native tryptophanfluorescence, and/or increase separation of differing ultravioletradiation signals. The radiation source may advantageously beincorporated into a computer-controlled light source and condenser lensassembly 56. The computer-controlled light source and condenser lensassembly 56 may further include condenser lens optics 24, 26 a lightdiffuser 28 and the radiation source 29.

In one example embodiment, the tube 22 is placed in a viewing areabetween two optically flat surfaces such as a standard microscope slide23A and a standard microscope coverslip 23B. The interstices between thetube 22 and the microscope slide 23A and coverslip 23B are filled withoptical gel 32 or an equivalent material such as inorganic and organicoils, having an index of refraction that also substantially matchesthose of the tube 22, and the microscope slide and coverslip. The tube22 itself may advantageously be coated with an oil of similar opticalproperties. The outer diameter of the tube 22 may be, for example about250 microns. Although not always shown in order to simplify the drawingsfor clarity, it will be understood that refractive index matchingmaterials are used to match optics in the various embodiments describedherein. A typical refraction index, n, matched to capillary tubing usedin an optical tomography system is about 1.48, for example, at 590 nm,but the dispersion curve moves sharply upward in the UV. Estimatedrefractive index of fused silica capillary tube is 1.51 at 250 nm, andtransmittance of DUV by UV grade fused silica is about 90%.

The biological object 1 may advantageously be selected from the groupconsisting of a cell, a live cell, a fixed cell, an unfixed cell, afrozen cell, a thawed cell, a desiccated cell, a cloned cell, a mobilecell, an immobilized cell, an encapsulated cell, a cell nucleus, cellparts, an organelle, a sub-cellular component, chromosomes, andequivalent materials. The optical tomographic imaging system 11 mayadvantageously employ illumination radiation having a frequency thatstimulates native fluorescence from the biological object, where thelight detectors and image processor further include modules formeasuring the stimulated fluorescence. The biological object iscontained in aqueous environment 2. The aqueous environment 2 comprisesphysiological buffered saline or other solutions as described below.

A beamsplitter 15 is positioned to split radiation transmitted throughthe biological object into at least two selected wavelengths. Thebeamsplitter may advantageously be selected from the group consisting ofa polarizing beam splitter, a Wollaston prism, a birefringent element, ahalf-silvered mirror, a 50/50 intensity beamsplitter, a dielectricoptically coated mirror, a pellicle film, a dichroic beamsplitter,mirror, prism, diffractive optical element, grating, and equivalents.The first light detector 10 is positioned to sense radiation transmittedthrough the biological object 1, the microscope objective 18, thebeamsplitter 15 and a first set of the optional filters 12A. Similarly,the second light detector 14 is positioned to sense radiationtransmitted through the biological object 1, the microscope objective18, the beamsplitter 15 and a second set of the optional filters 12B. Inone example, the first and second light detectors 10, 14 may eachparticularly include a pixel array detector sensitive to ultravioletlight, where each pixel array detector is selected to detect a differentone of the two selected wavelengths.

A computer 41 includes an image processor 40 coupled to receive datafrom the first and second light detectors 10, 14. A reconstructionmodule 42 is coupled to the image processor 40, where the reconstructionmodule processes the data to form a 3D image of the cell usingreconstruction algorithm techniques such as taught in Fauver '744 forexample. The image processor 40 transmits processed image data to the 3Dimage reconstruction module 42 which may advantageously be coupled to anoptical display 44 for operator viewing. User interface 46 can beprovided for operator control and information purposes. The userinterface 46 may be a GUI interface or the like coupled to the computer41.

In one example, the axial translation mechanism 34 comprises apiezoelectric transducer or equivalent device. A controller 35 linked tocontrol the piezoelectric transducer may advantageously be a computer,computer module or the like, where the piezoelectric transducer iscontrolled to axially move the objective lens 18.

In one example system, the optical tomographic imaging system 11 isconfigured through use of filters and radiation sources to image cellsusing wavelengths limited to between 240 nm and 300 nm. Radiationdetected by the first detector 10 may have wavelengths primarily in afirst range between 260 nm and 265 nm. Radiation detected by the seconddetector 14 may have wavelengths primarily in a second range between 280nm and 285 nm. The first range operates to enhance natural radiationabsorbance by DNA and RNA. The second range operates to enhance naturalradiation absorbance by protein. The first and second wavelength rangesmay be provided using a pair of radiation sources, each sourcetransmitting one of the two selected of wavelength ranges. One of thedetectors may be tuned to detect absorbance around 270 nm nearhydrophilic surfaces such as DNA and protein.

In one embodiment the radiation may be measured in time series usingtime to separate signals. The radiation source may be pulsed in a timeseries to cause pulsed excitation of the cells in order to increasesignal to noise, separating signals. For example, a radiation source at260 nm may be pulsed at a time, T₀, followed by a 280 nm pulse at T₁,followed in turn by one or more laser pulses at n subsequent timeincrements, T_(n), where n is any number denoting a subsequent point intime.

Alternatively, the native tryptophan fluorescence can be measured toobtain a secondary measure of protein and its confirmation andconstituents, such as amino acids. A third beam splitter would berequired unless time series illumination is used. In this alternativedesign, beamsplitter 15 would split all DUV light (240-300 nm) to theDUV light detector 14 while the lower frequency fluorescence signalwould be detected by a fluorescence light detector 10 (>300 nm).Operation of DUV light sources 30, 31 can be in time-series so radiationabsorbance primarily by nucleotides (260-265 nm) can be captured at timeT₀ while radiation absorbance primarily by amino acids (280-285 nm) canbe captured at time T₁ using the same detector 14. Discussion of filters12A, 12B is warranted in this example as the set before the fluorescencedetector will be the standard long-pass fluorescence emission filterswhile the set before the DUV detector will be DUV band pass filters orshort-pass fluorescence blocking filters.

In yet another example, laser light is incident at an oblique anglerelative to the objective lens optical axis, blocking the unscatteredlight and allowing dark-field measurement of the scattering profile athigher scattering angles. One example of the use of laser scattering atvisible wavelengths may be found in U.S. Pat. No. 6,741,730, issued May25, 2004 to Rahn, entitled “Method and Apparatus for Three-DimensionalImaging in the Fourier Domain,” which is incorporated herein byreference.

In still another example, laser illumination parallel to the opticalaxis is used. A disk of absorbing material is located in the back focalplane of the objective. The diameter of the absorber is only largeenough to block unscattered and very low-angle scattered light. Theresulting annular aperture permits a dark-field measurement of thescattering profile at higher scattering angles.

In still another example, live stain, either absorbance or fluorescence,in standard bright-field transmission mode (removing diffractionanalysis) or antibody/probe and nanoparticle is used in dark-fieldillumination mode for molecular specific labeling of proteins and/or DNAin the living cell.

In operation the image reconstruction module 42 determines a size of avoxel in the reconstructed 3D image. The reconstruction module 42 mayfurther include a module constructed in accordance with known softwareengineering techniques for measuring a concentration of moleculesabsorbing the radiation by measuring the absorbance per voxel.

In one useful embodiment, the optical tomographic imaging system 11lends itself nicely to DUV absorbance imaging. Using LEDs at 260 nm and280 nm with bandwidths of 20 nm allows for simple and robustinstrumentation without need for excitation filters. The condenseroptics 56 may include, for example, a DUV condenser lens (for example,model UV-Kond, from Zeiss, Germany) and objective lens 18 may comprise alens such as available from Zeiss, 100×, 1.25 NA, Ultrafluar, or acustom 265 nm objective lens, as available from Optics Technologies,Inc., Rochester, N.Y. To block the ambient and fluorescent light,filters 12A, 12B may include a band pass filter with a bandpass from 250nm to 290 nm as available from Chroma Technology Corp. or Omega Optical,both of Brattleboro, Vt., before light reaches the UV sensitive CCDcamera. Useful CCD cameras include CCD cameras from Sony Corporation ofJapan, the PhotonMax model camera from Princeton Instruments, Trenton,N.J., or devices from Sarnoff Imaging, Princeton, N.J.

Live cell imaging often requires the specimen stage and glycerol, oil,or water-immersion objective lens to be temperature controlled. Toconvert from 2D DUV imaging to 3D Cell-CT DUV imaging, the materialsmust be UV transparent for the short transmission distances (pathlengths) required for imaging isolated cells in a microcapillary tube of50 microns in diameter. For example, the cell medium should be aphysiological buffer solution that may have higher refractive index tohelp match to the cell plasma membrane. Additives to the aqueoussolution may include, but are not limited to, polyethylene glycol (PEG),glycerol, modified or derivative PEGs, and agarose gel. When the cellmedium cannot be well matched to the glass used for the microcapillarytube, then increasing the inner diameter may help reduce the degree ofrefraction at the inner tube wall. The refractive index should be ableto be matched well with the outer tube wall since no biocompatibilityneeds to be addressed. However, materials that do not fluoresce withinthe wavelength range of signal 250 nm-290 nm should be considered whenthe rotational joint is being selected.

Referring now to FIG. 2 an alternate example of system for 3D imaging ofcells in an optical tomography system with ultraviolet radiation using aUV camera and optional adaptive optics is schematically shown. Therequirement for live cell imaging imposes a restriction on the types ofaqueous and physiological buffer solutions and thus on the range ofrefractive index that can be used around the cell. This embedding mediumsurrounding the cell and within the tube is expected to have sufficientrefractive index mismatch with standard dry or oil immersion microscopeobjectives to cause aberrations in the resulting images. Compensationfor this index mismatch can be designed for a specified imaging depth ordistance from objective lens to cell that contains physiological buffer.However, even low-order spherical aberration varies with the variationin axial depth, so dynamic compensation of optical wavefront distortionis advantageous for microscopic imaging across axial depths. Thistechnique of dynamic distortion control or compensation is referred toas adaptive optics. The optical component used for such dynamicaberration compensation is often a spatial light modulator or adeformable membrane mirror. An adaptive reflection mirror is thepreferred component in a DUV microscope due to the non-optimaltransmission properties of DUV light through sophisticated opticalcomponents.

A system for 3D imaging of cells 200 includes several components thatare the same as or similar to those described above with respect toFIG. 1. As described above, a tube 22 is positioned relative to amicroscope objective 18 for viewing an object of interest 1. Asdescribed above, a microscope 16 includes an objective lens 18 and atube lens element 52. The microscope objective 18 is aligned along anoptical axis 202. In contrast to the system of FIG. 1, only a singleultraviolet (UV) camera 48 is used for acquiring images of the object ofinterest. The UV camera 48 is also aligned along the optical axis 202.Interposed between the UV camera 48 and the tube lens element 52 is afluorescence-blocking filter 50. As above, the fluorescence blockingfilter 50 is selected to block longer wavelength fluorescence and/orincrease separation of differing ultraviolet radiation signals.

The aqueous environment 2 and object of interest 1 may cause asufficiently large refractive index mismatch between microscopeobjective 18 and tube 22 and optical gel 32 or equivalent to necessitatethe use of an adaptive mirror 54 with associated adaptive optics (AO)controller 201 to reduce depth-dependent image aberrations. Thisadaptive optics component can be an optional element located between theradiation source 29, optical elements 27 and condenser lens 24. Whetherunpowered or energized at a constant wavefront compensation (2D)profile, the adaptive mirror 54 becomes a static 90-degree turn in theoptical system that may compensate for a single depth level.

As described above, images from the UV camera 48 are transmitted to theimage processor 40. The image processor transmits processed image datato the 3D image reconstruction module 42 which may advantageously becoupled to the optical display 44 for operator viewing if desired. Userinterface 46 is provided for operator control and information purposes.The user interface 46 may be a GUI interface or the like.

Referring now to FIG. 3, an embodiment of a temperature-controlledhousing for use in an optical tomography system is schematically shown.A temperature-controlled housing 300 contains an object of interest,such as a biological cell 1, or other biological material, is containedin a tube, capillary tube, or microcapillary tube 22, that is positionedrelative to a microscope objective 18. The microcapillary tube 22 isrotatable by a rotary motor 20 to allow controlled rotational motion 21of the cells 1 within the microcapillary tube 22. The cell 1 and gel 32can be advanced within the capillary tube 22 along the horizontal axisby positive pressure applied, for example, by a syringe 80. Anothermotor 34 controls vertical axial movement of the microscope objective18, and tube lens 52. The microcapillary tube 22 is encased withinoptical gel or refractive index matching medium 32 and is part of andatop of the sample-condenser light assembly 56.

A power amplifier 60 provides energy for the temperature controller 64that responds to at least one sensor 74 and that may be furtherregulated with computer and electronic input 78 to maintain the desiredtemperature within specified ranges, such as 5 to 39 degrees C. However,to maintain functions approaching physiological levels, a warm-bloodedanimal cell such as a human requires tight temperature control, i.e. 36degrees C. with range of +/−0.5 degrees C. Regulation of temperature aswell as microfluidic conditions facilitates keeping cells alive (i.e.especially labile normal or abnormal cells, pre-cancerous, cancerous,viral infected; or other pathogenic cell populations). In one example,three sensors 74 are positioned near the microscope head 16 and aboveand below the microcapillary tube 22. An optional internal fan 68 forair circulation is present in some embodiments to aid in temperaturecontrol. Peltier thermoelectric heaters/coolers 70 may be positioned inthroughout the system and may be positioned both above and below themicrocapillary tube 22 provide thermal energy for fine temperaturecontrol. Additional locations for Peltier heaters/coolers 70 may beadvantageous in specific embodiments. Alternatives to thermoelectricheater/coolers and fans are the options of temperature controlled watercirculator or equivalents around a chamber that encloses the microscope.In some embodiments temperatures of about 35 degrees C. to about 36degrees C. are used, in others higher or lower temperatures mayfacilitate study of specific biological processes or for use of specificreagents in living cells.

Having described the optical tomography system in detail above, adescription of the operation of the system will now be presented inorder to aid understanding of the disclosure. Biological objects 1, suchas living cells, are injected into the microcapillary tube 22 via thesyringe device 80 where pressurized capillary flow 84 moves thebiological objects 1 to a viewing window beneath the objective lens 18of the microscope 16. At least one radiation source 29 (e.g. DUV andvisible light) is positioned to illuminate a part of the microcapillarytube 22 that includes the biological objects 1. In some embodiments theradiation wavelengths of about 260 nm to about 280 nm are used. Theradiation passes through the light diffuser 28 and condenser lensassembly 24, 26, as part of the sample-condenser light assembly 56. Theintegrated sensors 74, temperature controller 64 and fan 68 maintain thetemperature to maintain and increase cell viability. The system allowsnumerous variations to study living cells under defined and controlledconditions.

The optical tomography system described above and elsewhere usestemperature control and microfluidics to maintain suitable conditionssuch that any living biological material may be examined including, butnot limited to, cells from humans, as well as cells from any otherspecies. The cells, or other biological material, flow through one ormore tubes (e.g. microcapillary tubes) to facilitate imaging. In someembodiments the microcapillary tube 22 comprises a straight tube of morethan one channel. It is recognized that the optical tomography systemmay be used to harvest cells or sub-cellular material in certainembodiments.

In some embodiments the system senses radiation including imagingsignals emanating from macromolecular complexes, nucleoprotein, DNA,RNA, or protein, comprised in living cells, or in some cases non-livingcells, or fragments thereof. Cells comprising component DNA, RNA, andprotein complexes may be treated with chemicals, biological agents,including, but not limited to biologically active molecules,nanoparticles, modified nanoparticles, microspheres, protein protocells,antibodies, biomarkers, cytokines, other nucleotides, other proteins, oralternately mechanically manipulated by micromanipulation or othertreatments (e.g. transfection reagents, viruses, liposomes, and likeagents) to alter or facilitate molecular uptake or affect other cellularprocesses during the imaging process. Biological or chemical agents maybe labeled or modified with chromophores and fluorophores. Embodimentsalso use nanoparticles that are modified by labeling with gold,colloidal gold, iron, and iron oxide, and like molecules that haveabsorption, fluorescence, and scattering properties acting as opticalcontrast mechanisms in the 3D image or diffraction pattern. Use ofnanoparticles and microspheres in addition to chromophores andfluorophores allows enhanced 3D contrast. For example, cells could betreated with agents that affect the cell cycle, cellulardifferentiation, infectivity, reduce or increase pathogenicity, or thecells can be further manipulated to alter sub-cellularcompartmentalization. The expression and display of cell surfacebiomarkers or chromatin or other cellular nucleoprotein ormacromolecular complexes could be examined during all or some of thesetreatments.

In certain embodiments the living cells or other biological material areilluminated with multiple wavelengths of radiation. In such cases, aplurality of pseudoprojection images of the cell, or other biologicalmaterial that are formed from the computer processing of input imagesmay be processed using ratio imaging techniques. In some embodiments theratio imaging includes images formed from radiation wavelengths of about260 nm to about 280 nm.

Alternately, in some cases, live cell staining techniques including, butnot limited to fluorescence and laser diffraction may be used toadvantage for obtaining images.

Referring now to FIG. 4, a side view of an example of a microfluidicscartridge 400 is schematically shown. A rotary motor 20 includes a shaft121 coupled to turn a belt 188, where a second end of the belt 188 iscoupled to rotate a microcapillary tube 22. The microfluidics cartridgeoperates with positive pressure and negative pressure to move the cellsin a raceway with a secondary channel to supply nutrients and oxygen,remove metabolic waste, and allow drugs to interact with cells inphysiological buffer (as best shown in FIG. 5). A bearing or frictionfit 92 allows the microcapillary tube 22 to rotate while an object, suchas a cell, passes through the tube. A microscope 16 including condenserillumination assembly 56 is positioned proximate the cartridge to viewthe object along the optical axis of the microscope 16.

Referring now to FIG. 5, a top view of an example of a microfluidicscartridge as used in a racetrack configuration for imaging cells isschematically shown. The microfluidics cartridge 400 is coupled in afluidic racetrack configuration 500. The racetrack configurationincludes an imaging area 116 along the optical axis of the objectivelens including an optical window. Also included is an entrance valve 96,an exit valve 124 and a first channel 502. The first channel 502 is influid communication with a secondary channel 504. The channels may bejoined, for example, with a semi-permeable membrane 104. The entireracetrack is maintained in a temperature controlled environment suchdescribed herein with respect to FIG. 3 using Peltier heater/coolerelements or equivalents.

Fresh nutrients, oxygen, buffer (pH, osmolarity, etc), optional drugsand the like as needed to maintain cell viability may be introducedthrough the secondary channel 504 as indicated by flow arrows 108.However, if microfluidic conditions are right, then the cells won't movelaterally, only axially through the first channel 502 while diffusionallows fresh nutrients such as O₂, buffer materials and metabolic wasteto move and thus mix along concentration gradients. In one example, thesemi-permeable membrane 104 may be replaced by a joined channel withnon-turbulent parallel flows allowing diffusion of small molecules andsolutions while maintaining cells within their original streamlines ofmicrofluidic flow. Shear stress within physiological range is possiblewith slow flow rates while channel geometry, fluid viscosity,temperature, and cell type also play a role.

In operation cells are injected through entrance valve 96 into themicrofluidics cartridge 400. A trough 100 serves as a housing for therotation motor and belt used to rotate the microcapillary tube 22 whilecells travel through the tube. Positive and negative pressure 120 isapplied to control pressurized flow 84 throughout the racetrack. Afterimaging, a an exit valve 124 can be used to direct selected cell 1 byflowing fluid into a discard channel or for harvesting the live cell.

The specimen being examined may be a biopsy from a fine needle aspirate(FNA). The resulting sample of live cells may be split into severaldifferent racetracks with separate entrance valves (not shown). Eachsub-sample being examined may be exposed to different drugs (such asdrug A, drug B, drug combination A+B, and control—no drug), and theresponse may be monitored as real-time feedback for the purpose ofpersonalized drug response for the patient.

In one example, the racetrack configuration is useful as a research/drugdiscovery instrument. In operation, live cells may be circulated in theracetrack while imaging in 3D. Each live cell in the sample may beexposed to a chemical and environmental protocol and small changes incellular response may be indicative of a desired cell type. Variationsis apoptosis, mitosis, necrosis, secretion, and other programmed cellresponses to stimuli can be measured at high sensitivity in real-time.When the live cells exhibit desired characteristics, the cells may beharvested. One such harvesting method is disclosed in co-pending USpatent application to Hayenga, entitled, “Cantilevered coaxial flowinjector apparatus and method for sorting particles,” and published onSep. 20, 2007 under publication number US 2007-0215528 A1, the fulldisclosure of which is incorporated herein by reference.

In some alternative embodiments, labeled nanoparticles like antibody/DNAlabeling of gold or nanospheres can be used with live cells to labelspecific proteins, chromatin, and DNA. For example, gold nanoparticlesor colloidal gold have both absorption and scattering contrast and arebiocompatible with living cells. Fluorescently-labeled nanospheres andmicrospheres can have absorption, fluorescence, and scattering asoptical contrast mechanisms in the 3D image or diffraction pattern.Using nanoparticles in addition to chromophores and fluorophores willallow a third contrast enhancement, which is scattering. A means forimaging the scatter signal as high contrast on a “black” background orfield is to illuminate with light that is incident at an angle ofincidence beyond that of the imaging objective lens, so only the signalscatter is collected. The image is analogous to that of fluorescenceimaging where the illumination photons are rejected from the finalimage. Live cell imaging in 2D using dark-field microscopy is beingconducted at Duke University, see, for example, Curry, A., Hwang, W. L.,and Wax, A. (2006), “Epi-illumination through the microscope objectiveapplied to dark-field imaging and microspectroscopy of nanoparticleinteraction with cells in culture,” Optics Express 14(14): 6535-6542.

Diffraction pattern measurement is a non-imaging technique that iscomplementary to the above imaging techniques which measure the spatialpattern in 3D of DNA, chromatin, proteins, and their specific labelingenhancements. Disease specific signatures of diffraction may be found atspecific spatial frequencies, which are measured at specific scatteringangles from the cell. Since the zero order light from the laser beam isorders of magnitude greater than the weakly scattered light from livecells, the technique of oblique illumination of the cell is proposed togreatly reduce this zero order light from reaching the optical detectoror camera. This technique is similar to dark-field microscopy usingnanoparticles as discussed above.

Examples of each of the techniques above may also be implemented ascombinations using some general concepts described below. However,laboratory implementation will most likely be done as examples of theindividual techniques for simplicity and lack of confounding variablesduring the development stage of live cell 3D imaging. Some examples ofcombining multiple imaging and measurement techniques are presentedbelow.

Referring now to FIG. 6, an optical tomography process includingseparate imaging stages along the same pathway is shown. Separateimaging stages may be processed along the same pathway, such as a singlemicrocapillary tube. For example, visible light diffraction analysis andcell counting 602 may be done at a first stage 611, followed by visiblelight imaging 604 at a second stage 612. In the case of imaging usinglive stains, 280 nm absorption imaging 606 may be conducted at a nextstage 613, followed by 260 nm absorption imaging 608 at a fourth stage614. For this example embodiment the cell should be aligned within thelimited field of view at each stage as the cell continuously moves downa single rotating capillary tube. The 280 nm absorption imaging includesilluminating the object 1 with DUV light at a first wavelength in therange of about 275 nm to 285 nm. The 260 nm absorption imaging includesilluminating the object 1 DUV light at a second wavelength in the rangeof about 255 nm to 265 nm.

In another example, a single imaging stage that combines one or moreimage contrast mechanisms, such as absorption at wavelengths of 260 nmand 280 nm, measuring DUV absorption and native fluorescence, ormeasuring absorption at more than two visible wavelengths for one ormore live stains. The components for combining optical imagingtechniques can use multiple optical components for beam splitting andcombining (dichroic or polarization beamsplitters) and possibly multiplecameras. Alternatively, a single camera and detection pathway can beused if the multiple excitation light sources are pulsed in time seriesor filter wheels or actual sources are physically moved or shuttered intime series. The single stage for imaging and measurement allow forstopped flow axial transport of the cells for precise alignment with thefield of view.

In yet another example, dark-field imaging of live-cell stain withnanoparticle scatterers may advantageously be combined with obliqueillumination of the cell with a laser for diffraction pattern analysis.This technique may be run at higher speeds and may be an initial stagebefore the slower and subsequent 3D imaging stage if initial resultswarrant a detailed 3D image of a particular cell.

In operation, the system provides an optical tomography processincluding separate imaging stages along the same pathway. A plurality ofbiological objects is transported along a pathway 25 to the first stage611. At least one object of the plurality of objects is illuminated withvisible light at the first stage to produce a diffraction pattern andthe diffraction pattern is sensed by a light sensor. Using a computerprogram or equivalent, the diffraction pattern is analyzed to produce adiffraction analysis. At the second stage 612 the at least one object 1is illuminated with visible light and the visible light emanating fromthe at least one object is sensed to produce a first plurality ofpseudoprojection images. At the third stage 613 the at least one object1 is illuminated with DUV light at a first wavelength and the DUV lightat a first wavelength emanating from the at least one object is sensedto produce a second plurality of pseudoprojection images. At the fourthstage the at least one object is illuminated with DUV light at a secondwavelength that is sensed to produce a third plurality ofpseudoprojection images. Based on features derived from the first,second and third pluralities of pseudoprojection images and thediffraction analysis a plurality of objects may be sorted or otherwiseclassified using a sorter 610. The sorter 610 may be any of many typesof conventional classifiers, usually embodied in software residing in acomputer such as a statistical sorter, adaptive classifier, neuralnetwork or equivalents.

The invention has been described herein in considerable detail in orderto comply with the Patent Statutes and to provide those skilled in theart with the information needed to apply the novel principles of thepresent invention, and to construct and use such exemplary andspecialized components as are required. However, it is to be understoodthat the invention may be carried out by specifically differentequipment, and devices, and that various modifications, both as to theequipment details and operating procedures, may be accomplished withoutdeparting from the true spirit and scope of the present invention.

1. A system for 3D imaging of live cells in an optical tomography systemcomprising: a microfluidics cartridge including a tube positionedrelative to a microscope objective, a conduit loop having a first portcoupled to an entrance valve, a second port coupled to an exit valve, asemi-permeable membrane portion, a rotating portion and an imagingwindow; where the rotating portion is mounted between a first fittingand a second fitting, where the first fitting couples the rotatingportion to the entrance valve and the second fitting couples therotating portion to the exit valve; a rotation mechanism attached to therotating portion; a microscope objective located to view objects throughthe imaging window; an axial translation mechanism coupled to themicroscope objective; a second conduit interfacing with thesemi-permeable membrane, where the second conduit carries nutrients intothe conduit loop and waste products out of the conduit loop; at leastone radiation source positioned to illuminate the imaging windowincluding a biological object held therein, where the at least oneradiation source generates radiation having a spectral bandwidth limitedto wavelengths between 150 nm and 390 nm; at least one sensor positionedto sense radiation transmitted through the biological object and themicroscope objective; an image processor coupled to receive data fromthe sensor; and a reconstruction module coupled to the image processor,where the reconstruction module processes the data to form a 3D image ofthe biological object.
 2. The system of claim 1 wherein the nutrientsinclude oxygen and a buffer.
 3. The system of claim 1 wherein the axialtranslation mechanism comprises a piezoelectric transducer.
 4. Thesystem of claim 1 wherein the at least one radiation source comprises acomputer-controlled light source and condenser lens assembly.
 5. Thesystem of claim 1 wherein a computer is linked to control thepiezoelectric transducer, where the piezoelectric transducer axiallymoves the objective lens so as to extend the depth of field of theobjective lens.
 6. The system of claim 1 wherein the spectral bandwidthhas wavelengths further limited to between 240 nm and 300 nm.
 7. Thesystem of claim 1 wherein the spectral bandwidth has wavelengths furtherlimited to between 260 nm and 265 nm.
 8. The system of claim 1 whereinthe spectral bandwidth has wavelengths further limited to between 280 nmand 285 nm.
 9. The system of claim 1, wherein the radiation stimulatesnative fluorescence from the live cell, further comprising measuring thestimulated fluorescence.
 10. The system of claim 1, wherein a size of avoxel in the reconstructed 3D image is known, the reconstruction meansfurther comprising a means for measuring a concentration of moleculesabsorbing the radiation by measuring the absorbance per voxel.
 11. Thesystem of claim 1 wherein the biological object includes a live cell.12. The system of claim 1 wherein the microfluidics cartridge iscontained in a temperature-controlled aqueous environment.
 13. Thesystem of claim 12 wherein the aqueous environment comprisesphysiological buffered saline.
 14. The system of claim 1 wherein thesensed radiation includes imaging signals emanating from DNA.
 15. Thesystem of claim 1 wherein the sensed radiation includes imaging signalsemanating from protein.
 16. The method of claim 1 wherein the sensedradiation includes imaging signals emanating from hydrophilic surfaces.17. The system of claim 1 wherein the generated radiation comprisesmultiple wavelengths.
 18. The system of claim 1 wherein thereconstruction module processes the data using ratio imaging.
 19. Thesystem of claim 18 wherein the ratio imaging includes images formed fromwavelengths ranging from 260 nm to 280 nm.
 20. The system of claim 1wherein the at least one radiation source comprises multiple sources forgenerating at least first and second selected wavelengths selected toenhance natural radiation absorbance by DNA.
 21. The system of claim 1wherein the spectral bandwidth is limited to wavelengths selected toenhance natural radiation absorbance by protein.
 22. The system of claim1 wherein the at least one radiation source comprises multiple sourcestransmitting at least two selected wavelengths that are pulsed in timeseries.
 23. The system of claim 1 wherein the at least one radiationsource comprises multiple sources transmitting at least two selectedwavelengths.
 24. The system of claim 1 wherein the sensor is anultraviolet pixel array detector.
 25. The system of claim 1 comprising abeamsplitter positioned to split radiation into at least two selectedwavelengths.
 26. The system of claim 25 wherein the beamsplitter isselected from the group consisting of a polarizing beam splitter, aWollaston prism, a birefringent element, a half-silvered mirror, a 50/50intensity beamsplitter, a dielectric optically coated mirror, a pelliclefilm and a dichroic mirrored prism.
 27. The system of claim 1 whereinthe at least one sensor comprises: a first light detector positioned toreceive light having a first wavelength; and a second light detectorsensitive to a second wavelength, the second light detector positionedto receive light having the second wavelength.
 28. The system of claim27 wherein the first light detector is sensitive to radiation having awavelength matching the natural absorbance of human DNA.
 29. The systemof claim 27 wherein the second light detector is sensitive to radiationhaving a wavelength matching the natural absorbance of protein.
 30. Thesystem of claim 27 wherein the first light detector is sensitive toradiation having a wavelength that includes imaging signals emanatingfrom hydrophilic surfaces.
 31. The system of claim 1 further comprisingat least one filter interposed between the at least one sensor and thetransmitted radiation.
 32. The system of claim 1 wherein themicrofluidics cartridge and second conduit are enclosed in atemperature-controlled environment.
 33. An optical tomography methodincluding separate imaging stages along the same pathway comprising:transporting a plurality of biological objects along a pathway to afirst stage; illuminating at least one object of the plurality ofobjects with visible light at the first stage to produce a diffractionpattern; sensing the diffraction pattern; analyzing the diffractionpattern to produce a diffraction analysis; illuminating the at least oneobject with visible light at a second stage; sensing visible lightemanating from the at least one object to produce a first plurality ofpseudoprojection images; illuminating the at least one object with DUVlight at a first wavelength at a third stage; sensing the DUV light at afirst wavelength emanating from the at least one object to produce asecond plurality of pseudoprojection images; illuminating the at leastone object with DUV light at a second wavelength at a fourth stage;sensing the DUV light at a second wavelength emanating from the at leastone object to produce a third plurality of pseudoprojection images;sorting the at least one object responsively to the first, second andthird pluralities of pseudoprojection images and the diffractionanalysis.
 34. The method of claim 33 further comprising counting thebiological objects.
 35. The method of claim 33 wherein the plurality ofbiological objects comprise live cells.
 36. The method of claim 33wherein the DUV light at a first wavelength is in the range of 255nm-265 nm.
 37. The method of claim 33 wherein the DUV light at a secondwavelength is in the range of 275 nm-285 nm.
 38. The method of claim 33wherein the diffraction pattern further comprises a dark-field imagingpattern.